转录因子AP-2α调控网络的研究

发布时间：2018-01-27 07:18

本文关键词： AP-2α miRNA 相互作用蛋白药物敏感性膀胱癌　出处：《湖南师范大学》2016年博士论文　论文类型：学位论文

【摘要】：膀胱癌是泌尿系统最常见的恶性肿瘤。对于膀胱癌患者的治疗,一般采用切除手术,必要时结合化疗或(和)放疗及辅助抗癌药物。尽管如此,有些患者的治疗效果仍然不是很好,其主要原因是我们对膀胱癌潜在致病机理了解的比较少。许多研究表明,转录因子AP-2α是一个癌症抑制因子。在膀胱癌、乳腺癌等癌症病人中转录因子AP-2α的表达水平与化疗敏感性正相关。本论文系统地研究AP-2α在膀胱癌中的作用及上下游调控网络。AP-2α主要作为转录因子发挥功能。为了研究AP-2α调控的下游靶基因,我们在缺失AP-2α表达的膀胱癌细胞系UM-UC-3中过表达AP-2α,然后进行转录组测序。一共鉴定到差异表达基因247个,其中27个上调,220个下调,然后对它们进行生物信息学分析来研究它们在膀胱癌中作用。同时,我们还用RT-PCR验证了4个潜在的AP-2α靶基因,发现1个表达上调,3个表达下调,这与转录组测序结果是一致的。为了研究AP-2α对miRNA的转录调控,我们在膀胱癌细胞系UM-UC-3中过表达AP-2α之后,进行了小RNA测序。一共鉴定到差异表达miRNA 41个,其中3个表达上调,38个表达下调。AP-2α的功能受其相互作用蛋白的影响。为了发现新的AP-2α相互作用蛋白,我们采用免疫共沉淀结合质谱技术来寻找新的AP-2α相互作用蛋白。首先我们对高表达AP-2α的正常膀胱上皮细胞系SV-HUC-1蛋白进行交联,然后用AP-2α抗体和对照抗体IgG进行免疫沉淀,差异条带通过质谱鉴定。一共鉴定到118个蛋白。同时,我们还对其中一个蛋白质Ran进行了荧光免疫共定位来验证其与AP-2α的相互作用,结果表明Ran确实与AP-2α存在相互作用。为了研究miRNA对AP-2α基因的调控,我们通过生物信息学分析调控AP-2α的miRNA。其中,miR-193a-5p能够结合到转录因子AP-2αmRNA的编码区。进一步研究发现,在膀胱癌细胞系UM-UC-3中抑制miR-193a-5p可以提高AP-2α的表达水平,而在正常膀胱上皮细胞系SV-HUC-1中过表达mi R-193a-5p则抑制AP-2α的表达。同时,我们的研究还发现mi R-193a-5p通过抑制AP-2α的表达来降低膀胱癌细胞对化疗药物顺铂的敏感性。本文系统分析了膀胱癌细胞中AP-2α调控的靶基因和miRNA、AP-2α相互作用蛋白及miRNA对AP-2α的调控。这些研究加深了我们对AP-2α功能的理解,为揭示膀胱癌的致病分子机理、寻找新的生物标记物奠定了基础。
[Abstract]:Bladder cancer is the most common malignant tumor in the urinary system. For patients with bladder cancer, resection surgery is commonly used, combined with chemotherapy or / and radiotherapy and adjuvant anticancer drugs when necessary. Some patients are still not very effective, the main reason is that we know less about the underlying pathogenesis of bladder cancer. Many studies show that. Transcription factor AP-2 伪 is a cancer suppressor in bladder cancer. The expression of transcription factor AP-2 伪 was positively correlated with chemosensitivity in patients with breast cancer and other cancers. In this paper, we systematically studied the role of AP-2 伪 in bladder cancer and the main regulatory network. AP-2 伪. To study the downstream target genes regulated by AP-2 伪. We overexpressed AP-2 伪 in bladder cancer cell line UM-UC-3 without AP-2 伪 expression, and then sequenced the transcription sequence. A total of 247 differentially expressed genes were identified, 27 of which were up-regulated. There were 220 down-regulated genes, and then bioinformatics analysis was performed to study their role in bladder cancer. At the same time, four potential AP-2 伪 target genes were identified by RT-PCR. One expression was up-regulated and three down-regulated, which was consistent with the results of transcriptome sequencing. In order to study the transcriptional regulation of AP-2 伪 on miRNA. After overexpression of AP-2 伪 in bladder cancer cell line UM-UC-3, we sequenced the small RNA. A total of 41 differentially expressed miRNA were identified, 3 of which were up-regulated. The function of down-regulated .AP-2 伪 expression was affected by its interaction protein. In order to find new AP-2 伪 interaction protein. We used co-immunoprecipitation and mass spectrometry to search for new AP-2 伪 interacting proteins. Firstly, we cross-linked the SV-HUC-1 protein of normal bladder epithelial cell line with high expression of AP-2 伪. Then the AP-2 伪 antibody and the control antibody IgG were used for immunoprecipitation, and the differential bands were identified by mass spectrometry. A total of 118 proteins were identified. One of the proteins, Ran, was also co-located by fluorescence immunoassay to verify its interaction with AP-2 伪. The results showed that Ran actually interacted with AP-2 伪. In order to study the regulation of AP-2 伪 gene by miRNA. We regulate the miRNAs of AP-2 伪 by bioinformatics analysis, in which miR-193a-5p can bind to the coding region of transcription factor AP-2 伪 mRNA. Inhibition of miR-193a-5p in bladder cancer cell line UM-UC-3 can increase the expression of AP-2 伪. The overexpression of mi R-193a-5p in normal bladder epithelial cell line SV-HUC-1 inhibited the expression of AP-2 伪. Our research also found that mi. R-193a-5p reduces the sensitivity of bladder cancer cells to cisplatin by inhibiting the expression of AP-2 伪. The target genes and miRN regulated by AP-2 伪 in bladder cancer cells were systematically analyzed. A. These studies have deepened our understanding of the function of AP-2 伪 and the regulation of AP-2 伪 by miRNA in order to elucidate the pathogenesis of bladder cancer. The search for new biomarkers laid the foundation.
【学位授予单位】：湖南师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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